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Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species - PubMed

  • ️Wed Jan 01 2014

Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species

Krishna K Gopaul et al. BMC Res Notes. 2014.

Abstract

Background: The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays.

Results: Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel.

Conclusion: The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

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Figures

Figure 1
Figure 1

Melt peaks of one HRM assay with titrations of target and non target DNA. The results of a titration range of 1 ng-100 fg genomic DNA from B. melitensis 16 M (red) and B. abortus 544 (blue) using the B. melitensis HRM assay and the negative first derivative melt peak data. As shown, whilst the peak size does vary with the concentration of DNA, there is clear differentiation between the two species.

Figure 2
Figure 2

Melt curves of one HRM assay with titrations of target and non target DNA. The results of a titration range of 1 ng-100 fg genomic DNA from B. melitensis 16 M (red) and B. abortus 544 (blue) using the B. melitensis HRM assay and the HRM melt curves directly. Curves move from right to left with decreasing DNA concentration. As shown, the curves generated by very low concentrations of B. abortus DNA are very close to the curves generated by high concentrations of B. melitensis DNA and could be misidentified through this association.

Figure 3
Figure 3

Determination of species identity based on melt curve kinetics. This figure gives an example of the diagnostic analysis of an unknown sample (green curve) comparing melt curves with curves generated in the quintuplex assay by representative strains of known Brucella species (grey curves). Species identification is based on “best match” with the curves in this case clearly indicating that the query strain is B. melitensis. Note that curves generated by B. microti isolates, not a target species for this assay, are shown to illustrate that a novel trace is generated for species out-with the target scope.

Figure 4
Figure 4

Melt curve kinetics for quintuplex target species. Melt profiles illustrating the unique HRM curves generated by the quintuplex for B. abortus (black), B. melitensis (green), B. ovis (light blue), B. suis (dark blue) and B. canis (purple). Each region of differentiation is highlighted.

Figure 5
Figure 5

Melt curve kinetics for quintuplex non target species. This figure illustrates how a Brucella isolate that is not a member of one of the five major species would be excluded by this assay. The curves in grey are traces from the five species targeted by the quintuplex whilst the traces in blue represent a non-target Brucella species (in this case B. microti). The blue traces are not congruent with any of the grey traces indicting that the sample tested represents a species not included in this assay.

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